Ivan Milenkovic

Ki67 index in intracranial ependymoma: a promising histopathological candidate biomarker

Aims:  The Ki67 tumour cell proliferation index is an independent prognostic factor in ependymoma patients. Essential prerequisites for validation of the Ki67 index as a histopathological biomarker are the reproducibility of this factor and its prognostic influence by different observers (proof of objective clinical and analytical performance). To this end, the aim was to analyse systematically inter‐ and intraobserver agreement and reproducibility of the prognostic impact of the Ki67 index in intracranial ependymoma.

Nigral burden of α‐synuclein correlates with striatal dopamine deficit

Aggregated α‐synuclein is the hallmark of Parkinsons disease (PD), diffuse Lewy body disease (DLBD), and multiple system atrophy (MSA). Physiologically, α‐synuclein ensures normal functions of dopamine transporter (DAT) and tyrosine hydoxylase. In α‐synucleinopathies, it accumulates in neuronal cytoplasm and neurites through several stages. It is unclear whether the accumulation of pathological α‐synuclein in the substantia nigra in PD correlates with the dopaminergic deficit in the striatal target. We evaluated the impact of the nigral burden of pathological α‐synuclein immunoreactivity in 27 α‐synucleinopathy brains by morphometric immunohistochemistry. DAT immunoreactivity in the striatum inversely correlates with the total α‐synuclein burden in the substantia nigra but not with cytoplasmic inclusion counts only. This result has implications for imaging, clinicopathological correlative studies, and staging of the disease process.

Distinct Patterns of Sirtuin Expression During Progression of Alzheimer’s Disease

Aging is one of the major risk factors for Alzheimer’s disease (AD). Sirtuins are associated with prolonged life span. To examine whether the expression levels of sirtuins associate with the progression of AD or not, we performed a comparative immunoblotting and immunohistochemical study of SIRT1, 3, and 5 in the entorhinal cortex and hippocampal subregions and white matter in 45 cases grouped according to Braak and Braak stages of neurofibrillary degeneration. In addition, we compared the expression levels with the local load of tau and amyloid-beta deposits, evaluated using morphometry. Our study revealed that (1) the neuronal subcellular redistribution of SIRT1 parallels the decrease in its expression, suggesting stepwise loss of neuroprotection dependent on the neuronal population; (2) in contrast to SIRT1 and 3, expression of SIRT5 increases during the progression of AD; (3) which might be related to its appearance in activated microglial cells. The complex patterns of the expression of sirtuins in relation to tissue damage should be taken into account when searching for therapies interacting with sirtuins.

mTOR Hyperactivation in down syndrome hippocampus appears early during development.

The mammalian target of rapamycin (mTOR) signaling pathway is a key developmental pathway involved in mechanisms underlying cellular aging and neurodegeneration. We hypothesized that its deregulation may occur during early brain development in patients with Down syndrome (DS). The expression patterns and cellular distribution of components of mTOR signaling (phosphorylated S6, phosphorylated S6 kinase, phosphorylated eukaryotic initiation factor 4E binding protein 1, and phosphorylated mTOR) were investigated in developing hippocampi from controls and patients with DS and from adults with DS and Alzheimer disease-associated pathology using immunocytochemistry. In control hippocampi, only phosphorylated S6 was detected prenatally (19-41 gestational weeks); it became undetectable 2 months postnatally. Increased expression of phosphorylated S6, phosphorylated S6 kinase, phosphorylated eukaryotic initiation factor 4E binding protein 1, and phosphorylated mTOR was observed in DS hippocampus compared with controls. Phosphorylated S6 and phosphorylated S6 kinase were detected prenatally and persisted throughout postnatal development. Prominent expression of mTOR components was observed in pyramidal neurons with granulovacuolar degeneration and in neurons containing neurofibrillary tangles in the hippocampi of DS subjects with Alzheimer disease pathology. These findings suggest that a dysregulated mTOR pathway may contribute to both early hippocampal developmental abnormalities and hippocampal functional impairment developing before neurodegeneration. Moreover, the expression patterns of mTOR components in adult DS hippocampus support its association with Alzheimer disease-related histopathologic changes.

Linking pathways in the developing and aging brain with neurodegeneration

The molecular and cellular mechanisms, which coordinate the critical stages of brain development to reach a normal structural organization with appropriate networks, are progressively being elucidated. Experimental and clinical studies provide evidence of the occurrence of developmental alterations induced by genetic or environmental factors leading to the formation of aberrant networks associated with learning disabilities. Moreover, evidence is accumulating that suggests that also late-onset neurological disorders, even Alzheimers disease, might be considered disorders of aberrant neural development with pathological changes that are set up at early stages of development before the appearance of the symptoms. Thus, evaluating proteins and pathways that are important in age-related neurodegeneration in the developing brain together with the characterization of mechanisms important during brain development with relevance to brain aging are of crucial importance. In the present review we focus on (1) aspects of neurogenesis with relevance to aging; (2) neurodegenerative disease (NDD)-associated proteins/pathways in the developing brain; and (3) further pathways of the developing or neurodegenerating brains that show commonalities. Elucidation of complex pathogenetic routes characterizing the earliest stage of the detrimental processes that result in pathological aging represents an essential first step toward a therapeutic intervention which is able to reverse these pathological processes and prevent the onset of the disease. Based on the shared features between pathways, we conclude that prevention of NDDs of the elderly might begin during the fetal and childhood life by providing the mothers and their children a healthy environment for the fetal and childhood development.

The Cell Adhesion Molecule Neuroplastin-65 Is a Novel Interaction Partner of γ-Aminobutyric Acid Type A Receptors

Background: Mechanisms of γ-aminobutyric acid type A (GABAA) receptor anchorage remain elusive. Results: The cell adhesion molecule neuroplastin-65 can be co-purified with GABAA receptors and co-localizes with α1, α2, and α5 but not α3 subunits. Conclusion: Neuroplastin-65 interacts with distinct receptor subtypes at synaptic or extra-synaptic sites. Significance: This interaction might contribute to a novel mechanism of GABAA receptor anchorage affecting receptor mobility and synaptic strength. γ-Aminobutyric acid type A (GABAA) receptors are pentameric ligand-gated ion channels that mediate fast inhibition in the central nervous system. Depending on their subunit composition, these receptors exhibit distinct pharmacological properties and differ in their ability to interact with proteins involved in receptor anchoring at synaptic or extra-synaptic sites. Whereas GABAA receptors containing α1, α2, or α3 subunits are mainly located synaptically where they interact with the submembranous scaffolding protein gephyrin, receptors containing α5 subunits are predominantly found extra-synaptically and seem to interact with radixin for anchorage. Neuroplastin is a cell adhesion molecule of the immunoglobulin superfamily that is involved in hippocampal synaptic plasticity. Our results reveal that neuroplastin and GABAA receptors can be co-purified from rat brain and exhibit a direct physical interaction as demonstrated by co-precipitation and Förster resonance energy transfer (FRET) analysis in a heterologous expression system. The brain-specific isoform neuroplastin-65 co-localizes with GABAA receptors as shown in brain sections as well as in neuronal cultures, and such complexes can either contain gephyrin or be devoid of gephyrin. Neuroplastin-65 specifically co-localizes with α1 or α2 but not with α3 subunits at GABAergic synapses. In addition, neuroplastin-65 also co-localizes with GABAA receptor α5 subunits at extra-synaptic sites. Down-regulation of neuroplastin-65 by shRNA causes a loss of GABAA receptor α2 subunits at GABAergic synapses. These results suggest that neuroplastin-65 can co-localize with a subset of GABAA receptor subtypes and might contribute to anchoring and/or confining GABAA receptors to particular synaptic or extra-synaptic sites, thus affecting receptor mobility and synaptic strength.

Endocannabinoids modulate cortical development by configuring Slit2/Robo1 signalling

Local environmental cues are indispensable for axonal growth and guidance during brain circuit formation. Here, we combine genetic and pharmacological tools, as well as systems neuroanatomy in human fetuses and mouse models, to study the role of endocannabinoid and Slit/Robo signaling in axonal growth. We show that excess 2-arachidonoylglycerol, an endocannabinoid affecting directional axonal growth, triggers corpus callosum enlargement due to errant CB1 cannabinoid receptor (CB1R)-containing corticofugal axon spreading. This phenotype mechanistically relies on the premature differentiation and end-feet proliferation of CB2R-expressing oligodendrocytes. We further show the dependence of both axonal Robo1 positioning and oligodendroglial Slit2 production on cell-type specific cannabinoid receptor activation. Accordingly, Robo1 and/or Slit2 manipulation limits endocannabinoid modulation of axon guidance. We conclude that endocannabinoids can configure focal Slit2/Robo1 signaling to modulate directional axonal growth, which may provide a basis for understanding impaired brain wiring associated with metabolic deficits and prenatal drug exposure.

The α1, α2, α3, and γ2 subunits of GABAA receptors show characteristic spatial and temporal expression patterns in rhombencephalic structures during normal human brain development.

γ‐Aminobutyric acid (GABA) is the most abundant inhibitory neurotransmitter in adult mammalian brain, mediating its actions chiefly via a pentameric chloride ion channel, the GABAA receptor. Nineteen different subunits (α1‐6, β1‐3, γ1‐3, δ, ε, π, θ, ρ1‐3) can give rise to multiple receptor subtypes that are the site of action of many clinically important drugs. In the developing brain, however, GABAA receptors mediate excitatory actions due to an increased chloride concentration within neurons and seem to control cell proliferation, migration, differentiation, synapse maturation, and cell death. Little is known about the distribution of single subunits in the human brain. Here we describe developmental changes in the immunohistochemical distribution of four subunits (α1, α2, α3, and γ2) in the human rhombencephalon. The γ2 was the most abundant subunit in all rhombencephalic structures during development and in adults, whereas α subunits showed a structure‐ and age‐characteristic distribution. The α1 was expressed prenatally in the molecular and Purkinje cell layer, but only postnatally in the granule cell layer and the dentate nucleus. Expression was completely absent in the inferior olivary nucleus. The α2 gradually increased during development, showing some layer specificity in the cerebellar cortex. The α3‐immunoreactivity in the cerebellar cortex was relatively weak, but it was abundantly observed in different cell populations in the subcortical cerebellar structures. Structure‐ and age‐characteristic colocalization between subunits during development suggests differences in GABAA receptor composition. Interestingly, subunit expression in several instances differed between human and rodent brain, underlining the importance of immunohistochemical studies in humans. J. Comp. Neurol. 524:1805–1824, 2016.

Patterns of Hippocampal Tau Pathology Differentiate Neurodegenerative Dementias

Background/Aims: Deposits of phosphorylated tau protein and convergence of pathology in the hippocampus are the hallmarks of neurodegenerative tauopathies. Thus we aimed to evaluate whether regional and cellular vulnerability patterns in the hippocampus distinguish tauopathies or are influenced by their concomitant presence. Methods: We created a heat map of phospho-tau (AT8) immunoreactivity patterns in 24 hippocampal subregions/layers in individuals with Alzheimers disease (AD)-related neurofibrillary degeneration (n = 40), Picks disease (n = 8), progressive supranuclear palsy (n = 7), corticobasal degeneration (n = 6), argyrophilic grain disease (AGD, n = 18), globular glial tauopathy (n = 5), and tau-astrogliopathy of the elderly (n = 10). AT8 immunoreactivity patterns were compared by mathematical analysis. Results: Our study reveals disease-specific hot spots and regional selective vulnerability for these disorders. The pattern of hippocampal AD-related tau pathology is strongly influenced by concomitant AGD. Mathematical analysis reveals that hippocampal involvement in primary tauopathies is distinguishable from early-stage AD-related neurofibrillary degeneration. Conclusion: Our data demonstrate disease-specific AT8 immunoreactivity patterns and hot spots in the hippocampus even in tauopathies, which primarily do not affect the hippocampus. These hot spots can be shifted to other regions by the co-occurrence of tauopathies like AGD. Our observations support the notion that globular glial tauopathies and tau-astrogliopathy of the elderly are distinct entities.

Shared and Distinct Patterns of Oligodendroglial Response in α-Synucleinopathies and Tauopathies

Pathological protein deposits in oligodendroglia are common but variable features of various neurodegenerative conditions. To evaluate oligodendrocyte response in neurodegenerative diseases (NDDs) with different extents of oligodendroglial protein deposition we performed immunostaining for tubulin polymerization-promoting protein p25&agr; (TPPP/p25&agr;), &agr;-synuclein (&agr;-syn), phospho-tau, ubiquitin, myelin basic protein, and the microglial marker HLA-DR. We investigated cases of multiple system atrophy ([MSA] n = 10), Lewy body disease ([LBD] n = 10), globular glial tauopathy ([GGT] n = 7) and progressive supranuclear palsy ([PSP] n = 10). Loss of nuclear TPPP/p25&agr; immunoreactivity correlated significantly with the degree of microglial reaction and loss of myelin basic prtein density as a marker of tract degeneration. This was more prominent in MSA and GGT, which, together with enlarged cytoplasmic TPPP/p25&agr; immunoreactivity and inclusion burden allowed these disorders to be grouped as predominant oligodendroglial proteinopathies. However, distinct features, ie more colocalization of &agr;-syn than tau with TPPP/p25&agr;, more obvious loss of oligodendrocyte density in MSA, but more prominent association of tau protein inclusions in GGT to loss of nuclear TPPP/p25&agr; immunoreactivity, were also recognized. In addition, we observed previously underappreciated oligodendroglial &agr;-synuclein pathology in the pallidothalamic tract in LBD. Our study demonstrates common and distinct aspects of oligodendroglial involvement in the pathogenesis of diverse NDDs.